Chitinase-3-like protein-1 at hospital admission predicts COVID-19 outcome: a prospective cohort study

Infectious and inflammatory stimuli elicit the generation of chitinase-3-like protein-1 (CHI3L1), involved in tissue damage, repair and remodeling. We evaluated whether plasma CHI3L1 at disease onset predicts clinical outcome of patients with Coronavirus 2019 (COVID-19) disease. Blood from 191 prospectively followed COVID-19 patients were collected at hospital admission between March 18th and May 5th, 2020. Plasma from 80 survivors was collected one month post-discharge. Forty age- and sex-matched healthy volunteers served as controls. Primary outcome was transfer to intensive care unit (ICU) or death. CHI3L1 was higher in COVID-19 patients than controls (p < 0.0001). Patients with unfavorable outcome (41 patients admitted to ICU, 47 died) had significantly higher CHI3L1 levels than non-ICU survivors (p < 0.0001). CHI3L1 levels abated in survivors one month post-discharge, regardless of initial disease severity (p < 0.0001), although remaining higher than controls (p < 0.05). Cox regression analysis revealed that CHI3L1 levels predict primary outcome independently of age, sex, comorbidities, degree of respiratory insufficiency and systemic inflammation or time from symptom onset to sampling (p < 0.0001). Kaplan–Meier curve analysis confirmed that patients with CHI3L1 levels above the median (361 ng/mL) had a poorer prognosis (log rank test, p < 0.0001). Plasma CHI3L1 is increased in COVID-19 patients and predicts adverse outcome.

Over one year after the onset of Coronavirus disease 2019 (COVID-19) pandemic, substantial progress has been made in patient management and in understanding disease mechanisms. Several signals have been proposed as putative players in the disease natural history [1][2][3][4][5][6][7][8] . As such, they may be interesting candidates as biomarkers of clinical outcome, helping inform management strategies and minimize the risk of disease progression. The ideal biomarkers should not just reflect the overall inflammatory burden but disclose the events responsible for adverse disease evolution, such as vascular inflammation and lung remodeling 9,10 . Moreover, since the interplay of antigen-presenting cells and T cells is crucial determinant of COVID-19 outcome, molecules involved in this process could be suitable candidates 11 . Chitinase-3 like-protein-1 (CHI3L1), a member of the glycoside hydrolase family 18, meets these requirements 12 . CHI3L1 binds to chitin, although being devoid of the ability to cleave the protein. It also binds to other substrates such as hyaluronic acid and heparin. Various signals that are activated in the early phases of COVID-19, including extracellular matrix (ECM) alterations, cell and tissue injury and response to cytokines and growth factors, elicit its synthesis by tissue cells and inflammatory leukocytes 12 . In turn, CHI3L1 stimulates the expression of angiotensin-converting enzyme 2 (ACE-2) and viral spike protein priming proteases in pulmonary epithelial and vascular cells 13 . ACE-2 is the functional receptor of SARS-CoV-2 and mediates viral entry through the viral spike protein 14 . After binding, the spike protein is cleaved by proteases into two subunits that mediate the fusion between viral envelope and cell plasma membrane 14 . ACE is expressed by several cells including enterocytes, renal tubular cells, cardiomyocytes and male reproductive and vascular cells 15 . ACE expression in the respiratory system is limited, in physiological conditions, to regions of the sinonasal  Fig. 1 panel B) Fig. 1 panel C), although remaining higher than HC (p < 0.05). CHI3L1 levels at admission were inversely correlated with respiratory function, as reflected by the PaO 2 /FiO 2 (R coefficient − 0.31, p < 0.0001, Fig. 2 panel A). On the contrary, CHI3L1 levels positively correlate with CRP levels (R coefficient 0.49, p < 0.0001, Fig. 2 panel B), NLR (R coefficient 0.34, p < 0.0001, Fig. 2 panel C) and LDH levels (R coefficient 0.29, p < 0.0001, Fig. 2 panel D).

CHI3L1 levels independently predict COVID-19 clinical outcomes. Multivariable Cox regression
analysis revealed that CHI3L1 plasma levels (ng/mL) at admission predict the risk of adverse outcome (transfer to ICU/death) independently of age, sex, comorbidities, degree of respiratory insufficiency and systemic inflammation (CRP) at admission, and time from symptom onset to blood draw (p < 0.01, Table 2). Risk of  www.nature.com/scientificreports/ adverse outcomes was increased by 0.5% per increase of 10 ng/mL of CHI3L1 plasma levels. Kaplan-Meier curve analyses confirmed that patients with plasma levels of CHI3L1 above the median value of 361 ng/mL had a significantly higher risk of adverse outcome (transfer to ICU or death, log rank test, p < 0.0001, Fig. 3) and of death (log rank test, p 0.0036, Supplementary Fig. 1) than those with lower levels. Multivariable Cox regression analyses were performed to investigate whether the ability of CHI3L1 to predict adverse outcome was independent of patient characteristic. CHI3L1 levels (ng/mL) predict transfer to ICU or death independent of HTN, COPD, CAD, DM, active neoplasia, CKD, NLR at admission, hospitalization, length of stay in hospital, steroid therapy and LMWH administration during hospitalization (Supplementary Table 1).

Discussion
We report that CHI3L1 accumulates early in COVID-19 patients. Patients with higher CHI3L1 levels had higher CRP levels and NLR values, reflecting a high inflammatory burden. These results agree well with those recently reported by 17 , who retrospectively compared serum levels of CHI3L1 in hospitalized COVID-19 patients, healthy subjects, patients with chronic obstructive pulmonary disease and with unrelated interstitial lung disease. We found that plasma levels of CHI3L1 were reduced in COVID-19 survivors four weeks after clinical remission, at a time when systemic inflammation had returned to baseline levels 25 . Interestingly, CHI3L1 levels at this point were still significantly higher than in healthy subjects, suggesting that stimuli sustaining CHI3L1 production are still present. The crosstalk between the ECM and CHI3L1 12 is intriguing since ECM remodeling occurs during acute COVID-19 26 , and may persist following disease resolution given the evidence of autoantibodies against ECM components in COVID-19 patients 27 .
High levels of CHI3L1 were associated with an increased risk of adverse outcome, including transfer to the ICU or mortality, independently of age, sex, comorbidities, degree of respiratory insufficiency and systemic inflammation at admission, and time from symptom onset to blood draw, all known to be associated with COVID-19 clinical outcome 28,29 . This observation indicates that the early generation of CHI3L1 is integral to the response initiated by the host recognition of SARS-CoV-2 and does not only reflect the magnitude of the inflammatory response or the clinical status. CHI3L1 is a recognized biomarker of kidney injury 30,31 , which makes it tempting to speculate that CHI3L1 levels may at least in part reflect kidney damage 32 . To verify this possibility, data on larger groups of patients are needed. Further studies are warranted to identify the stimulus involved in the persistent generation of CHI3L1 in patients with COVID-19 and the reasons of its preferential early accumulation in patients who will experience adverse outcomes. This might prove valuable to dissect at the molecular level the heterogeneity of the disease, possibly allowing a better understanding of the mechanisms responsible for COVID-19 complications.
Our study has limitations. First, the monocentric nature of the cohort implies that conclusions need to be further validated in larger independent cohorts. Second, although patients were prospectively followed, blood specimens at follow-up were available for only eighty of them. Third, a longer follow-up might add information, especially after stratifying survivors based on long-term sequelae 19,33 . In addition, the limited sample size may hamper the generalizability of results. Validation of our findings in larger cohorts support the routine use of CHI3L1 for prognostic and patient management purposes. Although quantification of CHI3L1 plasma levels is easy to perform through ELISA technique, it remains more expensive and time-consuming than measuring conventional biomarkers such as CRP, LDH or NLR, whose specificity for COVID-19 is however limited. Altogether, our data indicate that plasma levels of CHI3L1, a major player in the host response to inflammatory threats, are increased in patients with more severe COVID-19 patients and predicts adverse outcome independently of systemic inflammation and clinical indicators of severity. CHI3L1 may play a role in the progression of COVID-19 through mechanisms not directly related to the extent of the host inflammatory response. Further studies are needed to verify this claim. Kaplan-Meier curves depicting rates of adverse autcome (transfer to ICU or death) in patients with CHI3L1 levels below (low) or above (high) the median value of 361 ng/mL. Log rank test, p < 0.0001.